Standard and real time polymerase chain reaction (PCR) assays are limited by inhibitors found in many common sample types as well as components of the PCR assays themselves. Examples of PCR inhibitors include those found in commercial dyes used in PCR as well as those found in blood and soil.
Commonly used fluorescent dyes, such as SYBR Green, can significantly inhibit the Taq polymerase (GenBank Accession No. J04639; SEQ ID NO: 4) at concentrations higher than about 0.25-1×. This dye inhibition of Taq polymerase imposes limitations on the sensitivity and product specificity and may cause dye-dependant false negative results (see e.g., Monis et al. (2005) Anal Biochem 340, 24-34; Stubner (2002) J Microbiol Methods 50, 155-64; Nath et al. (2000) J Biochem Biophys Methods 42, 15-29; Gundry et al. (2003) Clin. Chem. 2003; 49:396-406).
The success and sensitivity of DNA detection in important clinical, diagnostic and forensic applications of PCR of blood specimens is limited by the presence of blood inhibitors of Taq polymerase, such as the heme, IgG fractions, and other blood components. Plain Taq enzyme can be completely inhibited between 0.004% and 0.2% blood (vol/vol) (see e.g., Al-Soud et al. (2000) J. Clin. Microbiol. 38, 4463-70; Al-Soud et al. (2000) J. Clin. Microbiol. 38, 345-50; Al-Soud et al. (1998) Environ. Microbiol. 64, 3748-53). In order to overcome this inhibition, high cost and additional labor-demanding methods are currently used to purify DNA from blood prior to PCR. Nevertheless, this inhibition is still a serious concern with many PCR-based human blood tests, since even after purifying DNA from the blood, traces of the PCR inhibitors can generate as high as 14% false negative results, as published for hepatitis B blood tests (Kramvis et al. (1996) J Clin Microbiology 34, 2731-2733).
Sensitive and precise PCR detection of microorganisms in soil is necessary, for example, in specific agricultural purposes, infectious disease control, and bioterrorism related pathogen tests. Direct extraction of total DNA from soil samples results in a co-extraction of humic acid, known as the most potent soil inhibitor to PCR analysis. Humic substances represent a mixture of partially characterized polyphenols that are produced during the decomposition of organic matter. Other inhibitory components include fulvic acid, polysaccharides and metal ions that can be present in varying concentrations in the soil samples (see e.g., Tsai et al. (1992) Environ. Microbiol. 58, 2292-2295; Watson et al (2000) Can. J. Microbiol. 46, 633-642; Yeates et al. (1998) Biol. Proced. Online 1, 40-47; LaMontagne et al. (2001) Journal of Microbiological Methods 49, 255-264).
A general technical problem with the soil samples is the high variation in the concentrations of various inhibitory substances, depending on the soil source, which may generate inconsistent results. This fact significantly complicates the development of standard DNA purification protocol for processing the samples before PCR. Unlike blood, crude soil extracts exhibit relatively little or no fluorescence quenching effect in real-time PCR.
U.S. patent application Ser. No. 11/005,559 (incorporated herein by reference in its entirety) describes blood-resistant mutants of Taq DNA polymerase, which can be used to amplify the DNA target directly in blood in standard, non-real-time PCR. Unlike existing protocols, this method requires no DNA purification steps prior to PCR, thus reducing the time and cost of important clinical blood tests. Today's PCR analyses, especially clinical and forensic analyses, increasingly utilize real-time PCR protocols, which allow accurate quantitation of the data. The blood, however, has a strong quenching effect on the fluorescence detected in real-time PCR, and such problem has previously been solved by using higher fluorescent dye concentrations.
Thus, there exists a need for inhibitor-resistant DNA polymerases and methods of their use for gene detection in standard and real-time PCR, both in the presence and absence of blood and soil in the samples, and for real-time assays, the presence of dye.